The in vitro fertilization of ova is undertaken in a variety of contexts. In human beings, a woman typically is treated with follicle stimulating hormone or is otherwise so treated as to stimulate superovulation. As a consequence, the numbers of follicles that are large and have the appearance of being ready to release an ovum at the proper time in the menstral cycle are increased. In any event, at the appropriate time the contents of at least some of the large follicles in the woman's ovaries are gently aspirated to remove the ova from the follicles, together with some of the follicular fluid in which they had been bathed. When it is desired to induce the woman to conceive, the ova are exposed to sperm in vitro and the resulting embryos are returned to the woman's uterus, where it is hoped implantation will occur and a pregnancy result.
An ovarian follicle is a constantly changing structure as it develops toward the point at which it may release an ovum. At any given point in that development, the ovarian follicle may become atretic. An atretic follicle subsides and eventually is entirely readsorbed within the tissue of the ovary, with no ovulation of the ovum. Ovarian follicles may be of any size at the point at which they become atretic. Thus, it is possible and in fact is normal for many follicles that are nearly at the point of releasing an ovum to become atretic. Without intervention, even such follicles as these subside with no release of a fertilizable ovum.
The change by which a follicle shifts from a developing follicle destined to release an ovum to an atretic follicle is an event not marked by an immediate visual change. Nevertheless, an ovum even if from a follicle that has only recently become atretic is unlikely to be fertilized when exposed to sperm. Thus, when several ova are removed from the ovaries of a woman, it is not possible by a visual examination to determine if a particular ovum was taken from a healthy follicle and is likely to undergo fertilization or if it is from an atretic follicle. As a partial consequence, when in vitro fertilization is being utilized as a means of obtaining a pregnancy in a woman, it is common procedure to remove several ova from the follicles of the woman, to attempt to fertilize them all, and then to return all of them to the woman's uterus.
The difficulty of reliably predicting the fertilizability of any given human ovum presents special problems because of ethical considerations. There is evidence suggesting that the ideal number of fertilized ova to be returned to a woman in order to achieve a pregnancy is approximately four. However, in many instances, as many as ten or more ova are harvested from the ovaries of a single woman. There is no simple and convenient conventional method by which it is possible to determine which ova out of the ten or more so removed are most likely to be capable of development. Available methods require expensive machinery, the employment of persons of high skill, and the use of radioactive substances.
In practice, it is common to attempt to fertilize all of the ova that have been removed from the ovaries. With all of the ova fertilized, the ethical need arises to reimplant all of them. It is deemed ethically repugnant to discard potentially viable, fertilized ova. But the result of reimplanting an increased number of ova is a reduced chance of ultimate success in achieving pregnancy.
It would be better to be able to discern those ova having a high probability of potential fertilization, to fertilize only them, and to implant only them. Ethical tensions would be reduced in that no fertilized ova would be abandoned. At the same time chances for a successful pregnancy would be increased by the use of the ova most likely to succeed in the numbers that have been shown to provide the best chance of success. It should be noted that, for reasons not clearly understood, it is most common that only one of the fertilized ova successfully implants and proceeds to develop, regardless of the number of ova reintroduced into the woman after fertilization.
In vitro fertilization is also used for purposes other than attempting to achieve pregnancy in humans. For example, in vitro fertilization has been used to achieve a bovine pregnancy. In addition, it is important to the bovine artificial insemination industry to assess the fertility of its bulls. Traditionally this has been done by attempting to fertilize large numbers of cows, carefully preserving data relating to numbers of inseminations necessary to achieve a pregnancy. A faster and less expensive alternative to this method is to collect large numbers of bovine ova at slaughter and attempt in vitro fertilization of the ova with semen taken from the bull to be tested. By this means, tests of the bull's ability to fertilize hundreds of ova may be completed in a few days' time at minimal cost and without putting valuable cows at risk. However, the results of such tests are suspect unless it can be determined if the ova to be used are themselves capable of being fertilized. As in the case of humans, discussed above, bovine ova from atretic follicles are unlikely to be fertilized in vitro, even by semen from the most fertile bull. It is not unusual for ova from atretic follicles to account for a third, a half, or even more of the ova harvested from an ovary. While it can be predicted that some of the ova will be from atretic follicles, it cannot be predicted, especially in relatively small samples, how many will be from atretic follicles. Consequently, the method of testing for bull fertility referred to remains inherently inaccurate without knowledge of the nature of the follicles from which ova used in the test have been taken.
Those skilled in the art are cognizant of methods for predicting the success of attempts to fertilize an ovum taken from a human follicle. The conventional method known to those skilled in the art involves an analysis of the follicular fluid in which the ovum has been bathed. See Ronald S. Carson et al., "Successful Fertilization of Human Oocytes in vitro: Concentration of Estradiol-17 beta, Progesterone, and Androstenedione in the Antral Fluid of Donor Follicles" (1982), J. Clin. Endocrinol. Metab., 55, 798-800. The method referred to involves a determination of the concentration of one or more of the steroids mentioned in the title of the article by Carson, et al. in fluid taken from human ovarian follicles from which ova have been aspirated. High concentrations of estradiol-17 beta are associated to a statistically significant extent with the achievement of fertilization in vitro, subsequent normal embryo growth, and pregnancy. High concentrations of progesterone also significantly correlate with successful in vitro fertilization and normal embryo growth. High concentrations of androstenedione are not predictive of successful in vitro fertilization but are predictive of normal embryo growth and subsequent pregnancy.
The concentrations of these steroids in the follicular fluid are very low, making analysis for them difficult. Typically, as in the article by Carson, et al. referred to above, concentrations must be determined by specific radioimmuno assays. A high degree of skill is involved in conducting such assays. The necessary equipment is expensive, and the facility undertaking the assay must be equipped and licensed to work with radio isotopes. As a consequence of all of these difficulties, analysis of follicular fluid for the steroids as a means for predicting the fertilizability of an ova has generally been limited to experimental situations or, in some instances, to human application in sophisticated medical facilities. The method is not available in realistic terms to the bovine artificial insemination industry, which has no commercially practical method for determining ova fertilizability. Likewise, many research facilities that are not equipped or licensed to undertake radioimmuno assays are cut off from research involving ova fertilizability.